Environmental stimuli, such as microorganisms and gluten, can lead to increased permeability of biological barriers and initiate significant pathological events in the intestine, brain, heart, and other organs. The pathological consequences of such stimuli include the development of inflammatory diseases. Such external stimuli are presumed to exert physiological effects on biological barriers, possibly through interaction with specific cell surface receptors. However, the mechanisms used remain unclear, and specific cell surface receptors have yet to be confirmed.
Many inflammatory diseases, including those that are understood to involve increased permeability of biological barriers, are thought to be autoimmune. Such diseases include celiac disease, rheumatoid arthritis, multiple sclerosis, immune-mediated or type 1 diabetes mellitus, inflammatory bowel diseases, systemic lupus erythematosus, psoriasis, scleroderma, necrotizing enterocolitis and autoimmune thyroid diseases. Prolonged inflammation is often associated with these diseases, although the inflammation is thought to be a sequela rather than a primary pathological insult.
Biological Barrier Dysfunction
Biological barrier function relies upon the structural and functional integrity of tight junctions (TJ), which are one of the hallmarks of absorptive and secretory epithelia. They act as a boundary that physically separates apical and basolateral compartments of epithelial cells, and they selectively regulate the passage of materials through the epithelia by controlling access to the space between the epithelial cells (the paracellular pathway). To meet the many diverse physiological and pathological challenges to which epithelia are subjected, the tight junctions must be capable of rapid, physiologic, reversible, transient, energy dependent, and coordinated responses that require the presence of a complex regulatory system. Examples of epithelia containing tight junctions include, but are not limited to, the intestines (particularly the small intestine), and the blood brain barrier.
In the absence of stimuli, tight junctions are closed restricting access to the paracellular pathway. In the presence of stimuli, the tight junctions are reversibly opened. Certain bacteria have been shown to have toxins that stimulate the opening of tight junctions. Vibrio cholerae infected with the filamentous bacteriophage CTXΦ, produces a toxin (zonula occludens toxin, ZOT) that has been shown to cause opening of tight junctions. It has been shown that 6 His-ΔG, an N-terminal deletion of ZOT in which the first 264 amino acids, have been deleted and replaced with a six histidine purification tag, retains the ability to open tight junctions.
Physiological changes in paracellular permeability, which are due to TJ regulation, can be measured as variations in transepithelial conductance. Such variations can usually be attributed to changes in paracellular permeability since the resistances of epithelial plasma membranes are relatively high. TJ represent the major barrier in the paracellular pathway, and the electrical resistance of epithelial tissues seems to depend on their integrity.
Environmental stimuli, including for example, microorganisms and gluten, can increase permeability of biological barriers as measured by a decrease in trans-epithelial electrical resistance (TEER) (ex vivo) or the Lactulose/mannitol test (in vivo). Such increases in barrier permeability are due primarily to TJ rearrangements, and they are believed to underlie many diseases including a large number of inflammatory conditions.
TJ dysfunction occurs in a variety of clinical conditions, including food allergies, infections of the gastrointestinal tract, autoimmune diseases, celiac disease and inflammatory bowel diseases. Healthy, mature gut mucosa with its intact tight junction serves as the main barrier to the passage of macromolecules. During the healthy state, small quantities of immunologically active antigens cross the gut host barrier. These antigens are absorbed across the mucosa through at least two pathways. Up to 90% of the absorbed proteins cross the intestinal barrier via the transcellular pathway, followed by lysosomal degradation that converts proteins into smaller, non-immunogenic peptides. These residual peptides are transported as intact proteins through the paracellular pathway, which mediates a subtle, but sophisticated, regulation of intercellular tight junction that leads to antigen tolerance.
In normal bowels, the immune reaction is regulated to maintain homeostasis of the gut. When TJ integrity is compromised, in premature infants or on exposure to environmental stimuli, radiation, chemotherapy, or toxins, a deleterious immune response to environmental antigens may develop. This response can result in autoimmune diseases and food allergies that lead to inflammation.
Inflammatory bowel disease (IBD) is a phrase used to describe an inappropriate immune response that occurs in the bowels of affected individuals. Two major types of IBD have been described: Crohn's disease and ulcerative colitis (UC). Both forms of IBD show abnormal profiles of T cell mediated immunity. In the gut of Crohn's disease a strong Th1 reaction is induced; the Th2 response is upregulated in the colon of UC.
The barrier function of the intestines is impaired in IBD. For example, Crohn's disease is associated with increased permeability of the intestinal barrier even in quiescent patients. A TNF-α-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn's disease and other inflammatory conditions. Increased intestinal permeability during episodes of active disease correlates with destruction or rearrangement of TJ protein complexes.
Examples of inflammatory diseases and disorders that may be treated using the instant invention include, for example, celiac disease, necrotizing enterocolitis, rheumatoid arthritis, multiple sclerosis, immune-mediated or type 1 diabetes mellitus, inflammatory bowel diseases (Crohn's disease and ulcerative colitis), systemic lupus erythematosus, psoriasis, scleroderma, and autoimmune thyroid diseases. Prolonged inflammation is often associated with these diseases, although the inflammation is thought to be a sequela rather than a primary pathological insult.
Other diseases and disorders associated with biological barrier dysfunction and which may be treated using the instant inventions include, for example, celiac disease, asthma, acute lung injury, acute respiratory distress syndrome, chronic obstructive pulmonary disease, inflammation (e.g., psoriasis and other inflammatory dermatoses), asthma, allergy, cell proliferative disorders (e.g., hyperproliferative skin disorders including skin cancer), metastasis of cancer cells, ion transport disorders such as magnesium transport defects in the kidney, and exposure to Clostridium perfringens enterotoxin (CPE). autoimmune encephalomyelitis, optic neuritis, progressive multifocal leukoencephalopathy (PML), primary biliary cirrhosis, IgA nephropathy, Wegener's granulomatosis, multiple sclerosis, scleroderma, systemic sclerosis, Hashimoto's thyroiditis (underactive thyroid), Graves' disease (overactive thyroid), autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, Devic's syndrome, Goodpasture's syndrome, Lambert-Eaton myasthenic syndrome (LEMS), autoimmune lymphproliferative syndrome (ALPS), paraneoplastic syndromes, polyglandular autoimmune syndromes (PGA), alopecia greata, gastrointestinal inflammation that gives rise to increased intestinal permeability, intestinal conditions that cause protein losing enteropathy, C. difficile infection, enterocolitis, shigellosis, viral gastroenteritis, parasite infestation, bacterial overgrowth, Whipple's disease, diseases with mucosal erosion or ulcerations, gastritis, gastric cancer, collagenous colitis, and mucosal diseases without ulceration, Menetrier's disease, eosinophilic gastroenteritis, diseases marked by lymphatic obstruction, congenital intestinal lymphangiectasia, sarcoidosis lymphoma, mesenteric tuberculosis, after surgical correction of congenital heart disease, and food allergies, primarily to milk.
Inflammation
Inflammation plays a central role in the pathology of disease conditions that adversely affect a considerable proportion of the population in developed countries. This process is mediated by cytokines, a system of polypeptides that enable one cell to signal to initiate events in another cell that initiate inflammatory sequelae. Normally, the system acts as part of a defensive reaction against infectious agents, harmful environmental agents, or malignantly transformed cells. But when inflammation exceeds the requirements of its defensive role, it can initiate adverse clinical effects, such as arthritis, septic shock, inflammatory bowel disease, and a range of other human disease conditions.
Immune cells such as monocytes and macrophages secrete cytokines including tumor necrosis factor-α (TNFα) and tumor necrosis factor-β (TNFβ) in response to endotoxin or other stimuli. Cells other than monocytes or macrophages also make cytokines including TNFα. For example, human non-monocytic tumor cell lines produce TNF. CD4+ and CD8+ peripheral blood T lymphocytes and some cultured T and B cell lines also produce TNFα. A large body of evidence associates cytokines such as TNFα with infections, immune disorders, neoplastic pathologies, autoimmune pathologies and graft-versus host pathologies.
Small-molecule antirheumatic drugs such as methotrexate and sulfasalazine are insufficient to control inflammation in about two-thirds of arthritis patients. New biological agents developed in the last decade have proved to be effective for a majority of patients unresponsive to traditional drugs. The target for such agents is often one of the cytokine pathways—either capturing the ligand conveying the signal from one cell to another, or blocking the receptor at the surface of the effector cell, preventing transduction of the cytokine signal, thereby forestalling the inflammatory events.
A leading biological agent for treating inflammatory conditions is Enbrel™ (Etanercept), marketed by Amgen Corp. It is a chimeric molecule comprising the extracellular portion of the human TNF receptor linked as a dimer to the IgG Fc region. The compound interferes with the binding of TNF to cell-surface TNF receptors—showing the importance of modulating the TNF pathway for clinical therapy of inflammatory conditions.
Other TNFα modulating agents currently licensed in the U.S. for treating inflammatory conditions include Cimzia™ (certolizumab pegol), a pegylated antibody fragment that binds to TNFα; Remicade™ (Infliximab), a chimeric antibody that binds TNFα; and Humira™ (adalimumab), a humanized anti-TNFα antibody.
Celiac Disease
Celiac disease (CD) is a chronic autoimmune disease that is HLA-DQ2/DQ8 haplotype restricted. Glutens, the major protein fraction of wheat, and related proteins in rye and barley are the triggering agents of the disease. Ingested gluten or its derivative fractions (gliadin and subunits) elicit a harmful T cell-mediated immune response after crossing the small bowel epithelial barrier, undergoing deamidation by tissue transglutaminase (tTG) and engaging class II MHC molecules.
While the earliest events leading to CD involve innate immune responses, evidence in the literature seems to suggest that a dysfunctional cross talk between innate and adaptive immunity is also an important pathogenic element in the autoimmune process of the disease. Under physiological circumstances, the intestinal epithelium, with its intact intercellular tight junctions (tj), serves as a key barrier to the passage of macromolecules such as gluten. When the integrity of the tj system is compromised, as in CD, a paracellular leak (“leaky gut”) and an inappropriate immune response to environmental antigens (i.e., gluten) may develop.
In celiac intestinal tissues and in in vitro, ex vivo, and in vivo animal experiments, gluten/gliadin causes a rapid increase in permeability in normal and diseased states. Animal models likewise have demonstrated the association of gluten, increased paracellular permeability and other autoimmune diseases, including type 1 diabetes (T1D).
AT-1001 is an orally administered octapeptide (Gly Gly Val Leu Val Gln Pro Gly (SEQ ID NO:1), that appears to inhibit gliadin-induced TJ disassembly and prevent the associated increase in paracellular permeability. Experiments with ex vivo human tissue and in mice demonstrate that AT-1001 blocks the peak of F-actin increment induced by gliadin and inhibits gliadin induced reduction in intestinal Rt (resistance).
There is a continuing need in the art for methods to treat inflammatory and autoimmune diseases as well as diseases associated with biological barrier dysfunction more effectively and to discover or identify drugs which are suitable for treating inflammatory and autoimmune diseases as well as diseases associated with biological barrier dysfunction.